Alfalfa (Medicago sativa L.) is considered to be the most important cultivated forage crop in the world (Hanson et al., 1988; Michaud et al., 1988) and is often referred to as "Queen of the forage crops" because it is widely grown, has a superb balance of vitamins and minerals, is high yielding, is an excellent source of biological nitrogen fixation, and it serves as an attractive nectar source for honeybees (Barnes et al., 1988; Smoliak and Bjorge, 1983). Alfalfa has been bred for years for both forage quality and plant performance. Although alfalfa and other leguminous forage crops are high in protein, these plants are deficient in the sulfur amino acids (S-amino acids), methionine and cysteine (Kaldy et al., 1979). It has been shown that wool growth in sheep is limited by the availability of S-amino acids. Similarly, milk production by dairy animals is affected by the deficiency of S-amino acids in plants. Efforts to use conventional plant breeding and cell selection techniques to increase the S-amino acid content of alfalfa have met with little or no success.
A genetic engineering approach to improve the amino acid balance of alfalfa and other forage crops would be to introduce into these plants genes encoding proteins high in methionine driven by a strong constitutive promoter or a leaf promoter. In order to significantly alter the amino acid balance of legume forage, the foreign proteins should contain about 15 to 25% of S-amino acids and constitute 5 to 10% of the total leaf protein. To achieve these levels of protein accumulation, one has to ensure not only maximum levels of transcription and translation of the gene but also the stability of the protein. In regard to forage crops for ruminant animals, the digestibility of S-amino acid containing proteins by the rumen bacteria and the stomach enzymes is also an extremely critical issue in regard to providing a suitable forage crop for ruminant animals, but is often overlooked. Thus, the S-amino acid rich protein should be relatively resistant to degradation in the rumen (first stomach) of the ruminant animals and should be assimilated in the lower gastrointestinal tract.
Most of the concerted efforts in regard to nutritional improvement in plants has focused on seed proteins. Since corn and other cereal crops are not easily transformable, most work directed to seed protein modification has involved testing stability of modified prolamine proteins in transgenic tobacco (Williamson et al., 1988; Ohtani et al., 1990) and Xenopus oocytes (Wallace et al. 1988). The synthesis of lysine containing .alpha. zeins was also analyzed in transgenic tobacco and petunia seeds (Williamson et al., 1988; Ohtani et al., 1990). Both the normal and modified protein were found to have a very short half-life.
Efforts to improve the S-amino acid content of legume seed proteins have included introducing a 45 bp oligonucleotide containing six methionine codons into the third exon of a .beta.-phaseolin gene. Transformants containing this modified gene showed that the high methionine phaseolin was synthesized at the same level as the normal protein, but was very unstable and was rapidly turned over (Hoffman et al., 1988). Introduction of the extra amino acids in the .beta.-phaseolin protein probably caused a distortion in its secondary structure making it more susceptible to proteolytic degradation. DeClercq et al. (1990), replaced a 23 amino acid coding segment between the sixth and seventh cysteine residues of Arabidopsis 2S albumin, with three different high methionine coding fragments. These modified Arabidopsis 2S genes were transformed into A. thaliana, B. napus and tobacco. There was some accumulation of the protein in the seeds but not as much as predicted. (Chrispeefs, M., personal communication). The gene of the 2S albumin of Brazil nut, which contains up to 19% methionine, and driven by the .beta.-phaseolin gene promoter, has been introduced into tobacco (Guerche et al., 1990), rape (Altenbach et al., 1992) and soybean (Pioneer Seed Co.). Recently, Saalbach et al. (1994) synthesized the 2S albumin gene and engineered it behind the CaMV 35S promoter. The gene, when introduced into tobacco and some grain legumes, showed the highest level of expression in the plant leaves and the protein was localized in vacuoles. However, the Brazil nut albumin protein is extremely allergenic and may not be acceptable for consumption.
One approach to increase the pools of particular amino acids in plants has been to introduce bacterial genes encoding for key regulatory enzymes in amino acid biosynthetic pathways in plants. A bacterial gene encoding for aspartate kinase which is desensitized to feedback inhibition by lysine and threonine was fused to the .beta.-phaseolin gene promoter and introduced into tobacco. The seeds of the transgenic tobacco showed increased levels of free threonine and methionine (Karchi et al., 1993; Galili, 1995).
Very little effort has been made with regards to improving forage crop protein quality. Schoeder et al., (1991) introduced the chicken ovalbumin gene (cDNA), driven by a CaMV 35S promoter, into alfalfa. The transgenic alfalfa plants, however, showed very low level accumulation of the protein in the leaves (0.005%). The basis for such a low abundance of this protein in the transgenic alfalfa leaves was not determined.
Some efforts to obtain alfalfa mutants that have larger free methionine levels have also been attempted at the University of Wisconsin. Cell lines with resistance to growth inhibition by an amino acid analogs reportedly produce higher than normal amounts of the corresponding natural amino acid. Hence, growth on specific amino acid analogs has been used as selection tool to select for plants accumulating high levels of a particular amino acid. Amino acid over-production is usually due to relaxed feedback control of an enzyme involved in its production (Malega, 1978). In an attempt to improve the methionine content of alfalfa, mutagenized suspension culture cells of alfalfa were selected for resistance to growth inhibition by a methionine analog (Reish et al., 1981). A few cell lines containing high methionine pools were obtained, however, regeneration of these cell lines did not produce plants with high methionine content (personal communication, Bingham, ET).
Zeins are a group of alcohol soluble proteins that are synthesized during endosperm development in corn and constitute 50% of the total protein in mature seeds (Lee et al., 1976). The zeins can be divided into four groups, the .alpha., .beta., .gamma. and .delta., based on their solubility (Larkins et al., 1989). The zeins can also be separated by size into groups. The .alpha. zeins, which is the most abundant class, are made up of the 22 kD and 19 kD zeins; the central region of these proteins consists of repetitive peptides of about 20 amino acid residues (Argos, 1982). The .beta. zeins comprise the 15 kD zein which contains less proline and glutamine than the .alpha. zeins. The .gamma. zeins include the 27 kD and 16 kD class and are very rich in proline (25%). The .delta. zeins are a relatively minor class consisting of the 10 kD zein (Kirihara et al., 1988). All the zein classes are structurally unique. The repeat regions in the .alpha. and .gamma. zeins probably have a major role in the packing of protein bodies. Zeins, in general, contain extremely low levels of the essential amino acids lysine, tryptophan and to a lesser extent methionine. The 15 kD and 10 kD zeins, however, are distinguished by their extremely high methionine content (10% and 22.5%, respectively) (Giannaza et al., 1977).
The zeins are synthesized on the rough endoplasmic reticulum (RER) and they aggregate into protein bodies directly in the RER (Larkins and Hurkman, 1978). Based on the analysis of the zein composition of developing protein bodies in corn endosperm, Lending and Larkins (1989), have proposed a descriptive model for the pattern of zein deposition during protein body formation in corn endosperm: The .beta. and .gamma. zeins are the first to start accumulating within the RER. Subsequently, .alpha. zeins begin to accumulate as locules within the .beta. and .gamma. zeins. With time, the a zein locules fuse and form a central core while the .beta. and .gamma. zeins form a continuous layer around the periphery of the protein body. In a separate study, Esen and Stetter (1992), demonstrated that the .delta. zein occurs throughout the core region of the protein body.
Mutations in maize affect the expression of the different zein genes. Changes in zein gene expression in turn have direct impact on the amino acid composition of the seeds. Seeds of plants homozygous for the recessive mutation opaque-2, have increased levels of lysine compared to the wild-type seeds (Misra et al., 1972). The increase in lysine is due to the reduced expression of the 22 kD .alpha. zeins (Langridge et al., 1983). The inbred line BSSS-53 has 30% higher level of seed methionine compared to other inbred lines. This increase in methionine content is because of a two-fold increase in the level of the 10 kD zein (Phillips and McClure 1985).
Proteins that accumulate in the endoplasmic reticulum are known to have the amino acid sequence Lys(his) Asp Glu Leu (K(H)DEL) near their carboxy terminal end which prevents them from exiting into the Golgi (Pelham, 1990). The zeins and other prolamines, however, lack this sequence. A cognate of the 70-kD heat shock protein, BiP which functions as a molecular chaperone has been shown to be involved in the formation of prolamine protein body formation in rice endosperm (Li et al., 1993b). The involvement of BiP in the formation of zein protein bodies is based on the fact that BiP accumulates to high levels in the ER and on the abnormal protein bodies of some of the zein regulatory mutants of corn (Boston et al., 1991; Zhang & Boston, 1992). Overall, however, the mechanisms of zein targeting and assembly in protein bodies are poorly understood and it is not known whether inter- and intra-molecular interactions play a key role in protein body formation. Abe et al. (1991), have suggested that the cytoskeleton plays a role in the biogenesis of zein protein bodies.
As can be understood from the above, there remains a need in the art for plants and forage crops that contain stable protein bodies that are high in S-amino acid content. The subject invention provides a novel and advantageous means for improving the forage quality of plants.